Microfluidic Mixing of Polyamine with Acrolein Enables the Detection of the [4+4] Polymerization of Intermediary Unsaturated Imines: The Properties of a Cytotoxic 1,5-Diazacyclooctane Hydrogel

Abstract

The [4+4] polymerization of an unsaturated imine, generated from the condensation of a polyamine and excess acrolein, was investigated. The polyamine was added by micropipet to acrolein, immediately yielding a mixture of the immiscible polymeric material. Microfluidic mixing was used to gradually form the soluble diazacyclooctane polymers. The polymerization reaction ultimately gave an insoluble cationic hydrogel that adhered strongly to anionic compounds on cell surfaces, including sialoglycan, and displayed a high cytotoxicity.

Because various organic transformations (e.g., protein bioconjugation) involve unsaturated imines as intermediates, they are crucial structural motifs for certain biological responses.[1] Although unsaturated imines can be synthesized via reactions of aldehydes with amines, N-alkyl unsaturated imines have not been as thoroughly investigated from a synthetic viewpoint as the corresponding imines bearing electron-withdrawing groups on the nitrogen.[2] N-Alkyl congeners are geometric isomers that are in equilibrium with their aldehyde precursors, and because N-alkyl congeners contain both electrophilic and nucleophilic centers, they are very reactive. For example, poly­merization of unsaturated imines is due to their uncontrollable reactivity. Controlling unsaturated imines may help to identify novel amino modifications of biological relevance[3] [4] [5] [6] because primary amines (e.g., lysines or ethanolamines) are common in biological systems. Primary amines readily react with unsaturated aldehydes, which are abundant in nature, yielding the corresponding N-alkyl unsaturated imines.

In our research program, which explores the novel reactivities of imines, we recently found by chance that the unsaturated N-alkylimines participated in the hitherto unknown ‘head-to-tail’ [4+4] dimerization in the presence of hydroxyl or amino groups on the N-alkyl imino nitrogen (Scheme [1, a]).[5] [6] The reaction readily provided eight-membered heterocycles, the 1,5-diazacyclooctanes, at micromolar concentrations. The nucleophilic hydroxyl or amino groups on the imino nitrogen (X = OH or RNH in Scheme [1, a]) accelerated and stabilized 1,5-diazacyclooctane formation.

The reactivity profiles of unsaturated N-alkylimines informed our studies, which revealed for the first time that polyamines react smoothly with acrolein to provide 1,5-diazacyclooctanes (1a or 1aa in Scheme [1, b]).[6] Considering that acrolein is produced as a polyamine metabolite by amine oxidase under oxidative stress conditions[7] [8] and that its production is involved in the progression of certain medical disorders,[9,10] our finding of the facile production of 1,5-diazacyclooctanes, a previously unrecognized acrolein-modified polyamine, suggests a new mechanism that could contribute to acrolein-mediated oxidative stress (Scheme [1, b]).[6] Extensive chemical and biological studies have indicated that an excess of acrolein produced under oxidative stress conditions or during the combustion of organic materials, for example, during smoking, could mediate a sequential [4+4] imino-polymerization reaction with polyamines to yield a very toxic diazacyclooctane hydrogel 1aa.[6] The production of diazacyclooctane polymers could further accelerate the oxidative stress processes.

This study examined the [4+4] polymerization process, which occurred under oxidative stress conditions. Our key insight was to mix the polyamine and acrolein in a microfluidic device, which allowed us to analyze the time-dependent polymerization reaction, which depended on the in-channel diffusion-mediated mixing of the reactants, using ordered NMR spectroscopy (DOSY) methods. The polymerization process ultimately gave a transparent cationic hydrogel that adhered strongly to the anionic sialoglycan, present on cell surfaces. We thus show how microfluidic mixing efficiently reduced the heat generated from biorelated exothermic reaction, which led to clear detection of the biologically relevant polymerization process. Our microfluidic investigation also enabled to analyze the important interaction of the diazacyclooctane polymers with the cell-surface constituents at the molecular level to rationalize the high toxicity to the cells.

The [4+4] polymerization process involved first mixing spermine (1) in PBS buffer (to yield a spermine concentration of a few mM in the cells)[9] with two equivalents acrolein in a conventional reaction flask using a micropipette. Within ten seconds, the reaction rapidly provided an insoluble brown-colored solid (Figure [1, a] and b), which was heterogeneously composed of polymers, as demonstrated by solid-phase NMR analysis. Acrolein is a highly reactive unsaturated aldehyde that reacts exothermically with the four nitrogen atoms in spermine under micropipetting conditions to produce a variety of polymeric products through conjugate addition, aminoacetal formation, and [4+4] cycloaddition reactions.[11] The reactive acrolein itself could also be polymerized in the presence of the amines. The slow addition of diluted acrolein in PBS (down to 100 μM) reduced the production of the insoluble material to some extent, but the ¹H NMR spectrum of the soluble fraction still detected large quantities of unreacted spermine.

‘Mixing artifacts’ of the flask procedures, especially associated with the exothermic reaction, were excluded, and the reactivity profiles in biological systems, for example in cells, were modeled by focusing on the use of a microfluidic apparatus. Innovative continuous-flow microreactors have been used to realize efficient mixing and rapid heat transfer in the context of organic synthesis reactions.[12] We succeeded in applying the advantageous features of microfluidic systems to the key but problematic organic reactions under conventional batch conditions, in particular, to bioactive natural product synthesis.[13] The advantages of microfluidic mixing were then effectively applied here by constructing the system shown in Figure [2], based on our previous experience. A PBS solution containing spermine and acrolein in different concentrations was mixed at room temperature using a Comet X-01 micromixer[14] with a channel width of ca. 500 μm (Figure [2]). The flow rate was set to 1.5 mL/min to facilitate the visualization of rapid micromixing in the fluidic system. The reaction mixture was allowed to flow for a few seconds through a reactor tube (Φ = 1.0 mm, l = 3 cm), after which it was introduced into a sample tube and subsequently allowed to polymerize.[15]

To our surprise, even a highly concentrated PBS solution of spermine and acrolein (100 mM) produced a transparent product solution, that is, low-molecular-weight, soluble diazacyclooctane polymers 1aa that eluted from the outlet of the microfluidic apparatus (Figure [1, c]).[16] This result clearly contrasted with the reaction products obtained by using micropipette mixing, which immediately gave an insoluble solid material consisting of a variety of polymer mixtures. The solution then gradually became cloudy over 10 hours. Direct DOSY measurements[17] of the [4+4] polymerization reaction, performed using deuterated PBS, as shown in Figure [3], indicated the production of the diazacyclooctane polymers 1aa with an average molecular weight of 6,000 after 5 hours and of 15,000 after 10 hours (Figure [3]). It should be noted that the integrated proton signals of the [4+4]-polymeric product were consistent with the exclusive consumption of spermine to proceed the [4+4]-polymerization reaction. Acrolein-derived imines, so as acrolein itself, generally could not be observed clearly by NMR in water. Acrolein and its imines are the smallest and most reactive unsaturated compounds, hence they are in rapid equilibrium with the corresponding acetals and/or hydrated derivatives in aqueous media. We propose that imine formation and [4+4]-cycloaddition reaction proceeded simultaneously under the equilibrium conditions, and they gradually developed the 1,5-diazacyclooctane polymers (Scheme [1, b]). Hence, the microfluidic system efficiently removed the heat generated during mixing between the polyamines and the reactive acrolein and enabled us to analyze the previously unrecognized [4+4]-polymerization reaction, by avoiding the rapid formation of insoluble polymeric products.

The [4+4]-polymerization reaction continued over a few days to produce, ultimately, an insoluble, transparent, highly cationic, and conformationally flexible material hydrogel 1aa that contained 16 water molecules per polymer (w/w; Figure [1, c]). Considering that the diazacyclooctane hydrogel was cationic in nature, its interactions with a representative anionic cell surface glycan, sialoglycan, were examined (Figure [4]). Fluorescence microscopy imaging analysis of the hydrogel 1aa treated with the fluorescein-labeled sialoglycopeptide 2 [18] clearly revealed fluorescein-derived green fluorescence on the surface of the hydrogel.[19]

We previously showed that cells rapidly adhere to the diazacyclooctane hydrogel 1aa and are immediately lysed, resulting in cell death.[6] The strong interactions between the hydrogel and the anionic sialoglycan molecule, as indicated in Figure [4], and the highly cytotoxic nature of the eight-membered diazaheterocycles, such as 1a, observed previously, could account for the cytotoxic properties of the diazaoctane hydrogel under oxidative stress conditions. Diazacyclooctane hydrogels, which are produced during cigarette smoking or under oxidative stress stimuli, strongly adhere to negatively charged cell surface molecules, including sialoglycans or proteoglycans. The oxidase-mediated production of large quantities of acrolein near cell adhesion regions on the diazacyclooctane polymeric materials[4] [6] may account for the high cellular toxicity of the hydrogel 1aa.

In conclusion, we used microfluidic mixing procedures to control the reaction between polyamine and acrolein. Efficient mixing and heat transfer in the microchannel enabled the monitoring of the previously unrecognized time-dependent [4+4] polymerization of the intermediary unsaturated diimine. Polymerization successfully produced the highly cationic hydrogel, which interacted strongly with the sialoglycan, a representative negatively charged cell-surface component. The high affinity toward the negatively charged molecules on the cell surface provides a rationale for the highly toxic properties of the previously unrecognized hydrogel under oxidative stress conditions. Thus, the microfluidic conditions may be used to mimic and reveal previously unrecognized organic transformations in biosystems.

Acknowledgment

We thank Dr. Hiroyuki Koshino and Dr. Takashi Nakamura at RIKEN for the NMR structural analysis. This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, No. 23681047, 25560410, and 26560438; by a Research Grant from the Mizutani Foundation for Glycoscience; by a MEXT Grant-in-Aid for Scientific Research on Innovative Areas ‘Chemical Biology of Natural Products: Target ID and Regulation of Bioactivity’ (No. 26102743); by a grant for Incentive Research Projects, RIKEN; and by an AstraZeneca R&D Grant. This work was also performed under the Russian Government Program of Competitive Growth of Kazan Federal University.

• Reference and Notes


For representative reviews, see:
• 1a Tanaka K, Fukase K, Katsumura S. Chem. Rec. 2010; 10: 119
  Search in Google Scholar
• 1b Tanaka K, Fukase K, Katsumura S. Synlett 2011; 2115
  Thieme Connect
• 2 For recent elegant examples in which the new reactivities of the N-alkyl unsaturated imines are explored, see: Shimizu M, Hachiya I, Mizota I. Chem. Commun. 2009; 874 ; and references cited therein
• 3 Tanaka K, Siwu ER. O, Hirosaki S, Iwata T, Matsumoto R, Kitagawa Y, Pradipta AR, Okamura M, Fukase K. Tetrahedron Lett. 2012; 53: 5899
  CrossrefSearch in Google Scholar
• 4 Tsutsui A, Tanaka K. Org. Biomol. Chem. 2013; 11: 7208
  CrossrefSearch in Google Scholar
• 5 Tanaka K, Matsumoto R, Pradipta AR, Kitagawa Y, Okumura M, Manabe Y, Fukase K. Synlett 2014; 25: 1026
  Thieme ConnectSearch in Google Scholar
• 6 Tsutsui A, Imamaki R, Kitazume S, Hanashima S, Yamaguchi Y, Kaneda M, Oishi S, Fujii N, Kurbangalieva A, Taniguchi N, Tanaka K. Org. Biomol. Chem. 2014; 12: 5151
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• 7 Kehrer JP, Biswal SS. Toxicol. Sci. 2000; 57: 6
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• 8a Alarcon RA. Arch. Biochem. Biophys. 1970; 137: 365
  CrossrefSearch in Google Scholar
• 8b Kimes BW, Morris DR. Biochim. Biophys. Acta 1971; 228: 223
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• 8c Houen G, Bock K, Jensen AL. Acta Chem. Scand. 1994; 48: 52
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• 9a Sharmin S, Sakata K, Kashiwagi K, Ueda S, Iwasaki S, Shirahata A, Igarashi K. Biochem. Biophys. Res. Commun. 2001; 282: 228
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• 9b Yoshida M, Tomitori H, Machi Y, Hagihara M, Higashi K, Goda H, Ohya T, Niitsu M, Kashiwagi K, Igarashi K. Biochem. Biophys. Res. Commun. 2009; 378: 313
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• 10a Uchida K, Kanematsu M, Morimitsu Y, Osawa T, Noguchi N, Niki E. J. Biol. Chem. 1998; 273: 16058
  CrossrefSearch in Google Scholar
• 10b Furuhata A, Ishii T, Kumazawa S, Yamada T, Nakayama T, Uchida K. J. Biol. Chem. 2003; 278: 48658
  CrossrefSearch in Google Scholar
• 10c Uchida K, Kanematsu M, Sakai K, Matsuda T, Hattori N, Mizuno Y, Suzuki D, Miyata T, Noguchi N, Niki F, Osawa T. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 4882
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• 10d Uchida K. Pharmacia 2012; 48: 26 ; and references cited therein
  Search in Google Scholar
• 11 Jianhua L, Chun R, Zhu Y, Wenfang S. J. Polym. Sci., Part A: Polym. Chem. 2007; 45: 699
  CrossrefSearch in Google Scholar

For pioneering reviews, see:
• 12a Microreaction Technology . Ehrfeld W. Springer; Berlin: 1998
  CrossrefSearch in Google Scholar
• 12b Mycrosystem Technology in Chemistry and Life Sciences. Manz A, Becker H. Springer; Berlin: 1998
  CrossrefSearch in Google Scholar
• 12c Ehrfeld W, Hessel V, Lowe H. Microreactors . Wiley-VCH; Weinheim: 2000
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• 12d Hessel V, Hardt S, Lowe H. Chemical Micro Process Engineering . Wiley-VCH; Weinheim: 2004
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• 12e Yoshida J.-I, Suga S, Nagaki A. J. Synth. Org. Chem. Jpn. 2005; 63: 511
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• 13a Tanaka K, Fukase K. Org. Process Res. Dev. 2009; 13: 983
  CrossrefSearch in Google Scholar
• 13b Tanaka K, Fukase K. Beilstein J. Org. Chem. 2009; 5: No. 40
  Search in Google Scholar
• 14 Comet X-01 micromixer: http://homepage3.nifty.com/techno-applications/ or E-mail: yukio-matsubara@nifty.com.
• 15 The [4+4] Polymerization of the Spermine/Polyamine-Derived Diimine under Microfluidic Conditions A solution of spermine (150 mg, 741 μmol) in PBS (2.0 mL) was injected, in advance, into the micromixer, a Comet X-01, using a syringe pump at a flow rate of 1.5 mL/min. A solution of acrolein (100 μL, 741 μmol) dissolved in PBS (2.0 mL) was then injected into the micromixer using another syringe pump at a same flow rate. The reaction was mixed at r.t. The reaction mixture was allowed to flow at r.t. for a few seconds through a Teflon tube reactor (Φ = 1.0 mm, l = 3.0 m) and was then introduced into a flask and left for 2 d at this temperature. A deuterated PBS buffer was used in the procedure described above to analyze the polymerization reaction over time using NMR methods. The PBS buffer was freeze-dried twice from D₂O prior to use.
• 16 When the reaction mixture was eluted from the microfluidic apparatus, a complex mixture of smaller polymerized diazacyclooctane fragments with molecular weights of 1,000–3,0000 were detected.
• 17 Cohen Y, Avram L, Frish L. Angew. Chem. Int. Ed. 2005; 44: 520
  CrossrefSearch in Google Scholar
• 18 Tanaka K, Nakamoto Y, Siwu ER. O, Pradipta AR, Morimoto K, Fujiwara T, Yoshida S, Hosoya T, Tamura Y, Hirai G, Sodeoka M, Fukase K. Org. Biomol. Chem. 2013; 11: 7326
  CrossrefSearch in Google Scholar
• 19 Interactions between the 1,5-Diazacyclooctane Hydrogel and Sialoglycan 2 The 1,5-diazaoctane hydrogel 1aa (2.0 mg) was treated with an aqueous solution of sialoglycan 2 (10^–5 M), and the mixture was allowed to incubate for 3.5 h at r.t. The gel was washed with H₂O (3×) and further soaked in H₂O for 1 h. The resulting gel was analyzed by OLYMPUS fluorescence microscopy, IX71-23FL/DIC.

• Reference and Notes


For representative reviews, see:
• 1a Tanaka K, Fukase K, Katsumura S. Chem. Rec. 2010; 10: 119
  Search in Google Scholar
• 1b Tanaka K, Fukase K, Katsumura S. Synlett 2011; 2115
  Thieme Connect
• 2 For recent elegant examples in which the new reactivities of the N-alkyl unsaturated imines are explored, see: Shimizu M, Hachiya I, Mizota I. Chem. Commun. 2009; 874 ; and references cited therein
• 3 Tanaka K, Siwu ER. O, Hirosaki S, Iwata T, Matsumoto R, Kitagawa Y, Pradipta AR, Okamura M, Fukase K. Tetrahedron Lett. 2012; 53: 5899
  CrossrefSearch in Google Scholar
• 4 Tsutsui A, Tanaka K. Org. Biomol. Chem. 2013; 11: 7208
  CrossrefSearch in Google Scholar
• 5 Tanaka K, Matsumoto R, Pradipta AR, Kitagawa Y, Okumura M, Manabe Y, Fukase K. Synlett 2014; 25: 1026
  Thieme ConnectSearch in Google Scholar
• 6 Tsutsui A, Imamaki R, Kitazume S, Hanashima S, Yamaguchi Y, Kaneda M, Oishi S, Fujii N, Kurbangalieva A, Taniguchi N, Tanaka K. Org. Biomol. Chem. 2014; 12: 5151
  CrossrefSearch in Google Scholar
• 7 Kehrer JP, Biswal SS. Toxicol. Sci. 2000; 57: 6
  CrossrefSearch in Google Scholar
• 8a Alarcon RA. Arch. Biochem. Biophys. 1970; 137: 365
  CrossrefSearch in Google Scholar
• 8b Kimes BW, Morris DR. Biochim. Biophys. Acta 1971; 228: 223
  CrossrefSearch in Google Scholar
• 8c Houen G, Bock K, Jensen AL. Acta Chem. Scand. 1994; 48: 52
  CrossrefSearch in Google Scholar
• 9a Sharmin S, Sakata K, Kashiwagi K, Ueda S, Iwasaki S, Shirahata A, Igarashi K. Biochem. Biophys. Res. Commun. 2001; 282: 228
  CrossrefSearch in Google Scholar
• 9b Yoshida M, Tomitori H, Machi Y, Hagihara M, Higashi K, Goda H, Ohya T, Niitsu M, Kashiwagi K, Igarashi K. Biochem. Biophys. Res. Commun. 2009; 378: 313
  CrossrefSearch in Google Scholar
• 10a Uchida K, Kanematsu M, Morimitsu Y, Osawa T, Noguchi N, Niki E. J. Biol. Chem. 1998; 273: 16058
  CrossrefSearch in Google Scholar
• 10b Furuhata A, Ishii T, Kumazawa S, Yamada T, Nakayama T, Uchida K. J. Biol. Chem. 2003; 278: 48658
  CrossrefSearch in Google Scholar
• 10c Uchida K, Kanematsu M, Sakai K, Matsuda T, Hattori N, Mizuno Y, Suzuki D, Miyata T, Noguchi N, Niki F, Osawa T. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 4882
  CrossrefSearch in Google Scholar
• 10d Uchida K. Pharmacia 2012; 48: 26 ; and references cited therein
  Search in Google Scholar
• 11 Jianhua L, Chun R, Zhu Y, Wenfang S. J. Polym. Sci., Part A: Polym. Chem. 2007; 45: 699
  CrossrefSearch in Google Scholar

For pioneering reviews, see:
• 12a Microreaction Technology . Ehrfeld W. Springer; Berlin: 1998
  CrossrefSearch in Google Scholar
• 12b Mycrosystem Technology in Chemistry and Life Sciences. Manz A, Becker H. Springer; Berlin: 1998
  CrossrefSearch in Google Scholar
• 12c Ehrfeld W, Hessel V, Lowe H. Microreactors . Wiley-VCH; Weinheim: 2000
  CrossrefSearch in Google Scholar
• 12d Hessel V, Hardt S, Lowe H. Chemical Micro Process Engineering . Wiley-VCH; Weinheim: 2004
  CrossrefSearch in Google Scholar
• 12e Yoshida J.-I, Suga S, Nagaki A. J. Synth. Org. Chem. Jpn. 2005; 63: 511
  CrossrefSearch in Google Scholar
• 13a Tanaka K, Fukase K. Org. Process Res. Dev. 2009; 13: 983
  CrossrefSearch in Google Scholar
• 13b Tanaka K, Fukase K. Beilstein J. Org. Chem. 2009; 5: No. 40
  Search in Google Scholar
• 14 Comet X-01 micromixer: http://homepage3.nifty.com/techno-applications/ or E-mail: yukio-matsubara@nifty.com.
• 15 The [4+4] Polymerization of the Spermine/Polyamine-Derived Diimine under Microfluidic Conditions A solution of spermine (150 mg, 741 μmol) in PBS (2.0 mL) was injected, in advance, into the micromixer, a Comet X-01, using a syringe pump at a flow rate of 1.5 mL/min. A solution of acrolein (100 μL, 741 μmol) dissolved in PBS (2.0 mL) was then injected into the micromixer using another syringe pump at a same flow rate. The reaction was mixed at r.t. The reaction mixture was allowed to flow at r.t. for a few seconds through a Teflon tube reactor (Φ = 1.0 mm, l = 3.0 m) and was then introduced into a flask and left for 2 d at this temperature. A deuterated PBS buffer was used in the procedure described above to analyze the polymerization reaction over time using NMR methods. The PBS buffer was freeze-dried twice from D₂O prior to use.
• 16 When the reaction mixture was eluted from the microfluidic apparatus, a complex mixture of smaller polymerized diazacyclooctane fragments with molecular weights of 1,000–3,0000 were detected.
• 17 Cohen Y, Avram L, Frish L. Angew. Chem. Int. Ed. 2005; 44: 520
  CrossrefSearch in Google Scholar
• 18 Tanaka K, Nakamoto Y, Siwu ER. O, Pradipta AR, Morimoto K, Fujiwara T, Yoshida S, Hosoya T, Tamura Y, Hirai G, Sodeoka M, Fukase K. Org. Biomol. Chem. 2013; 11: 7326
  CrossrefSearch in Google Scholar
• 19 Interactions between the 1,5-Diazacyclooctane Hydrogel and Sialoglycan 2 The 1,5-diazaoctane hydrogel 1aa (2.0 mg) was treated with an aqueous solution of sialoglycan 2 (10^–5 M), and the mixture was allowed to incubate for 3.5 h at r.t. The gel was washed with H₂O (3×) and further soaked in H₂O for 1 h. The resulting gel was analyzed by OLYMPUS fluorescence microscopy, IX71-23FL/DIC.